ABSTRACT This is a competitive renewal of our R37 MERIT (2009-2019), which aims to define mechanisms of Pulmonary Arterial Hypertension (PAH). Our work led to new knowledge, shared resources and methods, including a critical advance in harvest and culture of human pulmonary artery endothelial cells (PAEC). We identified loss of nitric oxide (NO) production, and mechanisms: (i) phosphorylation inactivation of endothelial NO synthase (eNOS), and (ii) decreased eNOS substrate arginine (arg) availability related to increased mitochondrial arginase 2 (ARG2). We discovered that high-altitude natives, who avoid high-altitude hypoxic pulmonary hypertension (HAPH), have adaptations that increase arg, NO, and decrease ARG2. In parallel, we found that PAH PAEC have less mitochondrial respiration than control cells, and a shift to aerobic glycolysis. In vivo, lung and cardiac glucose uptake in PAH patients measured by 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) was higher than controls. In preliminary data, patients with the highest FDG uptake have the lowest arg levels and most severe disease, suggesting that arg metabolic fate impacts PAH beyond just the loss of vasodilator NO. We show that mitochondrial arg metabolism is interconnected to bioenergetics, including fuel dependency of tricarboxylic acid cycle (TCA) and mitochondrial respiration. In murine studies, Arg2 knockout (Arg2KO) have greater capacity for mitochondrial respiration and less cardiac glucose uptake than wildtype (WT), and lack the hypoxia-induced increases of pulmonary pressures and erythropoietin (Epo) found in WT. Thus, we hypothesize that mitochondrial arg metabolism via ARG2 is interconnected to abnormalities of TCA cycle and bioenergetics, and promotes pathologic proliferation of PAEC, development of PAH and right ventricular (RV) dysfunction. Aim 1 identifies disease metabolic mechanisms and pathways that participate in pathologic endothelial functions and PAH pathophysiology. To find pathways associated with PAH, we perform differential expression (RNA, protein, metabolites) and integrative network analyses of PAH and control PAEC, then measure cell bioenergetics and functions after blocking pathways enriched in PAH (Aim1A). PAH patients, dichotomized into low or high cardiac FDG uptake groups, are compared with controls using metabolomic and transcriptomic analyses to uncover pathways in vivo, then followed longitudinally to determine if death, transplant, and/or worsening RV systolic pressure (RVSP) is greater in the high uptake group (Aim1B). Aim 2 determines if decreasing mitochondrial arg metabolism is protective against hypoxia-associated PH. Metabolomic profiles of high-altitude Amhara, who have high NO, low arginase, and resist HAPH, are compared to their low-altitude counterparts and PAH patients, to identify protective pathways in relation to quantitative traits, such as Epo and RVSP (Aim 2A). In mechanistic studies, we determine if Arg2KO have bioenergetic changes as compared to WT, and if Arg2KO are protected from hypoxia-induced elevations in Epo and pulmonary pressures (Aim 2B). These studies will provide new metabolic understanding of PAH, and offer new potential therapeutic targets.